EFFECTS OF SODIUM CHLORIDE AND POLYETHYLENE GLYCOL ON THE WATER RELATIONS, GROWTH, AND MORPHOLOGY OF CITRUS ROOTSTOCK SEEDLINGS By MONGI ZEKRI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1987 In the name of God, Most Gracious, Most Merciful. "It is He Who has let free the two bodies of flowing water: one palatable and sweet and the other salt and bitter: yet has He made a barrier between them, a partition that is forbidden to be passed." Glorious Quran Sura XXV (Furquan), or The Criterion Verse #53 In the name of God Most Gracious, Most Merciful. "It is He Who sendeth down rain from the skies: with it We produce vegetation of all kinds: from some We produce green (crops), out of which we produce grain, heaped up (at harvest); out of the date-palm and its sheaths (or spathes) (come) clusters of dates hanging low and near: and (then there are) gardens of grapes, and olives, and pomegranates, each similar (in kind) yet different (in variety): when they begin to bear fruit, feast your eyes with the fruit and the ripeness thereof. Behold! in these things there are signs for people who believe. Yet they make the Jinns equals with God, though God did create the Jinns; and they falsely, having no knowledge, attribute to Him sons and daughters. Praise and glory be to Him! (for He is) above what they attribute to Him! To him is due the primal origin of the heavens and the earth: how can He have a son when He hath no consort? He created all things, and He hath full knowledge of all things. That is God, your Lord! There is no god but He, The Creator of all things: then worship ye Him: and He hath power to dispose of all affairs." Glorious Quran Sura VI (An'am), or Cattle Verses #99-102 ACKNOWLEDGMENTS The author expresses his deepest appreciation to his wife, Leila, for her assistance, encouragement, and patience. He also wishes to express his sincere gratitude to his mother and to all the family in Tunisia for their patience and understanding through the years the author was away from home. The author expresses his profound gratitude to Dr. L.R. Parsons, chairman of the supervisory committee, for his valuable advice and helpful suggestions in the course of conducting the research and in the preparation of the manuscript. Sincere thanks re extended to Dr. R. C. J. Koo and to Dr. W. S. Castle for their advice and for providing greenhouse space. A special debt of gratitude is acknowledged to Dr. D. L. Myhre and to Dr. A. G. Smajstrla for their helpful suggestions and comments and for kindly serving on the supervisory committee. The author is also grateful to Dr. J. P. Syvertsen and Mr. M. L. Smith, Jr., for providing equipment and for the use of their laboratory facilities. The author's most sincere gratitude is extended to the coordinators of the Tunisia Agricultural Technology Transfer Project for continuous encouragement and financial support. TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES ix ABSTRACT xii INTRODUCTION 1 LITERATURE REVIEW 3 Salts 3 Mechanisms of Salt Tolerance in Plants 3 Mechanisms of Salt Injury 4 Osmotic Effect 4 Ion Toxic Effect 5 Nutritional Imbalance 6 Plant Responses to Salinity 7 Salinity and Citrus 8 Citrus Salinity Research 8 Citrus Tolerance to Salinity 11 Scion 11 Rootstock 11 Salt exclusion 12 Ion concentration 12 Citrus Responses to Saline Conditions 13 Photosynthesis 13 Yield 14 Leaf injury 14 Salinity and high water table 15 Irrigation 15 Reducing Salt Damage 17 Role of Calcium 17 Genetic Improvement 18 MATERIALS AND METHODS 20 General Procedures 20 Experiment 1: Effects of NaCl and PEG on the Root Conductivity and Leaf Ion Content of Seedlings of 7 Citrus Rootstocks 21 Experiment 2: Water Relations of Sour Orange and Cleopatra Mandarin Seedlings under NaCl and PEG Stresses 26 Page Experiment 3: Fibrous Root Density and Distribution of Sour Orange Seedlings under NaCl and PEG Stresses 28 Experiment 4: Response of Split-Root Sour Orange Seedlings to Salinity 29 Experiment 5: Effects of Calcium on Sour Orange Seedlings Grown under Saline Conditions 32 RESULTS 34 Experiment 1: Effects of NaCl and PEG on the Root Conductivity and Leaf Ion Content of Seedlings of 7 Citrus Rootstocks 34 Experiment 2: Water Relations of Sour Orange and Cleopatra Mandarin Seedlings under NaCl and PEG Stresses 49 Experiment 3: Fibrous Root Density and Distribution of Sour Orange Seedlings under NaCl and PEG Stresses 57 Experiment 4: Response of Split-Root Sour Orange Seedlings to Salinity 65 Experiment 5: Effects of Calcium on Sour Orange Seedlings Grown under Saline Conditions 73 Comparison of Citrus Seedling Responses to NaCl and PEG Treatments 79 DISCUSSION 82 Leaf Ion Content and Salinity Tolerance 82 Rootstock Tolerance 82 Ion Exclusion and Accumulation 83 Leaf Ion Content and Ion Toxicity 84 Importance of Calcium under Saline Conditions 85 Physiological Effects of NaCl and PEG 86 Effect of NaCl on Root Conductivity 86 Effect of PEG on Root Conductivity 87 Effect of NaCl on Stomatal Conductance 88 Effect of PEG on Stomatal Conductance 89 Effect of NaCl and PEG on Chlorophyll 89 Effect of NaCl on Leaf Thickness and Succulence .... 90 Growth of Citrus Rootstock Seedlings under NaCl and PEG Stresses 90 Relationship of Leaf Damage Symptoms to Growth Reduction 91 Root Growth and Distribution under NaCl and PEG Stresses 91 Effects of Non-Uniform Salinity and Water Stress .... 92 Comparative Effects Between NaCl and PEG 93 SUMMARY AND CONCLUSIONS 95 APPENDIX 100 LITERATURE CITED 116 BIOGRAPHICAL SKETCH 132 LIST OF TABLES Table Page 1. Salt treatments and chemical properties of the different salt treatments 33 2. Shoot dry weight of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 35 3. Root dry weight of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 36 4. Specific fibrous root weight of seedlings of 7 rootstocks grown under different NaCl concentrations . . 38 5. Root length, root conductivity, water flow rate, and osmotic potential of root exudate of seedlings of 7 rootstocks under non-stressed conditions 40 6. Visible injury in seedlings of 7 rootstocks after 5 months of NaCl treatments 42 7. Leaf sodium content of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 44 8. Leaf chloride content of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 45 9. Ion exclusion and accumulation in leaves of citrus rootstock seedlings 47 10. Leaf calcium content of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 48 11. Monthly new flush growth--area/leaf — of sour orange seedlings 50 Table Page 12. Monthly new flush growth-leaf number--of sour orange seedlings 51 13. Leaf succulence of seedlings of 2 rootstocks grown for 6 months under different NaCl and PEG concentrations 54 14. Total chlorophyll of seedlings of 2 rootstocks grown for 6 months under different NaCl and PEG concentrations 55 15. Fibrous root length in the 3 compartments of the root boxes for seedlings under different NaCl and PEG concentrations 63 16. Shoot and root dry weight of split-root sour orange seedlings under NaCl and PEG stresses 66 17. Midday leaf water, osmotic, and turgor potentials of split-root sour orange seedlings under NaCl and PEG stresses 70 18. Midday stomatal conductance and transpiration of split-root sour orange seedlings under NaCl and PEG stresses 71 19. Root and shoot dry weight of sour orange seedlings under different salt treatments 75 20. Total plant dry weight and leaf succulence of sour orange seedlings under different salt treatments .... 76 21. Leaf mineral analysis of sour orange seedlings under different salt treatments 77 22. Summary of citrus rootstock responses to NaCl and PEG as compared to a no salt control 80 23. Shoot root ratio of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 100 24. Total plant dry weight of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 101 Table Page 25. Stem cross sectional area of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 102 26. Leaf magnesium content of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 103 27. Leaf potassium content of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 104 28. Leaf phosphorus content of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 105 29. Leaf zinc content of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 106 30. Leaf manganese content of seedlings of 7 rootstocks grown for 5 months under different NaCl and PEG concentrations 107 31. Seedling height of seedlings of 2 rootstocks grown for 6 months under different NaCl and PEG concentrations 108 32. Total leaf area of seedlings of 2 rootstocks grown for 6 months under different NaCl and PEG concentrations 109 33. Specific leaf weight of seedlings of 2 rootstocks grown for 6 months under different NaCl and PEG concentrations 110 LIST OF FIGURES Figure 1. Osmotic potential versus NaCl concentration as determined by vapor pressure (VPD) and freezing point depression (FPD) 22 2. Osmotic potential versus PEG concentration as determined by vapor pressure (VPD) and freezing point depression (FPD) 23 3. Sour orange seedlings with a split-root system 30 A. Effect of 3 NaCl concentrations on the total fibrous root length, root hydraulic conductivity, and water flow rate for seedlings of 7 citrus rootstocks 37 5. Relationship between root hydraulic conductivity and specific root weight of seedlings of 7 citrus rootstocks under non-stressed conditions Al 6. Effect of NaCl at an osmotic potential of -0.35 MPa on the 7 rootstocks after 5 months of salinity treatments A3 7. Relationship between water flow rate and osmotic potential of root exudate of sour orange and Cleopatra mandarin seedlings 53 8. Relationship between midday stomatal conductance and root conductivity of sour orange and Cleopatra mandarin seedlings 56 9. Midday stomatal conductance of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG ... 58 10. Relationship of time of day to stomatal conductance of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG during 2 consecutive days .... 59 Figure 11. Growth of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG 60 12. Fibrous root length of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG 61 13. Fluctuations in shoot and root growth of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG ... 62 14. Root density and distribution of sour orange seedlings growing in root boxes under non-stressed (NS) and stressed (NaCl, PEG) conditions 64 15. Root development of split-root sour ornage seedlings under uniform and non-uniform NaCl and PEG stress 67 16. Leaf water, osmotic, and turgor potential of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with NaCl added to both root halves 69 17. Relationship between transpiration and stomatal conductance of sour orange seedlings 72 18. Cross sections of sour orange leaves 74 19. Sour orange leaves from non-stressed (control) and stressed (NaCl, PEG) seedlings 81 20. Effect of 3 NaCl concentrations on the osmotic potential of root exudate collected from seedlings of 7 citrus rootstocks Ill 21. Relationship of time of day to stomatal conductance of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl during 3 consecutive days 112 22. Relationship of time of day to stomatal conductance of Cleopatra mandarin seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl during 3 consecutive days 113 Figure Page 23. Relationship of time of day to stomatal conductance of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added PEG during 3 consecutive days 114 24. Relationship of time of day to stomatal conductance of Cleopatra mandarin seedlings irrigated with nutrient solution containing no salt (NS) or with added PEG during 3 consecutive days H5 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF SODIUM CHLORIDE AND POLYETHYLENE GLYCOL ON THE WATER RELATIONS, GROWTH, AND MORPHOLOGY OF CITRUS ROOTSTOCK SEEDLINGS By MONGI ZEKRI December 1987 Chairman: Dr. Lawrence R. Parsons Major Department: Horticultural Science (Fruit Crops) The effects of sodium chloride (NaCl) and polyethylene glycol (PEG) on the growth, water relations, and leaf mineral content of citrus rootstocks were investigated. Significant growth reduction and physiological disturbances occurred even at NaCl and PEG concentrations of -0.10 MPa. Growth reduction and physiological changes were found to precede visible damage. Growth was reduced up to 30% without being accompanied by visible leaf injury symptoms. Leaf burn symptoms developed only after a threshold value of chloride accumulation (1%) was reached. Leaf burn symptoms developed too slowly to accurately evaluate salt damage. Root conductivity correlated better with salinity tolerance among rootstocks than did total fibrous root length. Sodium chloride usually caused less damage than PEG. Unlike PEG, NaCl significantly increased leaf thickness and succulence along with leaf sodium and chloride concentrations, but reduced calcium and zinc contents in the leaves. Both NaCl and PEG reduced leaf magnesium and potassium contents but increased leaf phosphorus and manganese contents. Differences in sodium and chloride exclusion capacities among rootstocks were found. Sour orange, rough lemon, and Milam were sodium and chloride accumulators. Poncirus trifoliata, Swingle citrumelo, and Carrizo citrange were sodium excluders but chloride accumulators. Cleopatra mandarin was a chloride excluder but a sodium accumulator. Differences in NaCl sensitivity among rootstocks were also found. Cleopatra mandarin and sour orange were the least sensitive, Milam and Poncirus trifoliata were the most sensitive, and rough lemon, Swingle citrumelo, and Carrizo citrange were intermediate in sensitivity. Cleopatra mandarin tolerated high concentrations of NaCl by partial exclusion of chloride while sour orange tolerated NaCl even though it accumulated sodium and chloride in its leaves. Sour orange might have the ability to compartmentalize these ions and exclude them from the cytoplasm where they may inhibit metabolic processes. Seedlings receiving NaCl or PEG produced small and shallow root systems with the majority of the roots occurring in the top layer. Addition of calcium sulfate to saline irrigation water was found to be beneficial in overcoming the detrimental effects of NaCl on citrus. The split-root experiment showed that citrus could withstand substantial amounts of stress as long as half of the root system was growing in a non-stressed environment. INTRODUCTION It is well established that salt can impair the performance of many agricultural plants. Salts present in the soil and irrigation water are a serious problem for commercial agriculture particularly in arid and semi-arid regions. However, the potential for salinity damage also exists in humid climates. Controlling or reducing salt injury is usually achieved either through soil management practices and irrigation with good quality water or by combining these practices with the use of salt-tolerant plants. Citrus is a fruit crop of international significance. It is grown in over 50 countries and ranks among the top 3 tree fruit crops in world production. In certain areas where citrus is grown, salinity is already a problem of some importance. In other areas, the future of citriculture is threatened by salinity largely because agriculture is being forced to use lower quality land and water for irrigation. In agricultural areas with salinity problems, citrus is particularly vulnerable because there is relatively little salt tolerance in the genus. Salinity studies have shown that among species, cultivars, and various selections, only 2 roots tocks, Cleopatra mandarin and Rangpur lime, have a limited capacity to tolerate certain salts. However, rootstocks are usually selected for other attributes such as yield and fruit quality. Rootstocks deficient in these characteristics are not likely to be commercially used despite favorable salt tolerance. 1 2 In Florida, there are many citrus plantings located in coastal areas where saline water is being used for irrigation. Citrus planting in these and other southern Florida areas has been accelerated by extensive freeze damage in more northern areas. These changes in the citrus industry, as well as the diseases triteza and blight, have greatly affected interest in rootstock characteristics including salt tolerance. In the past, virtually all evaluations of citrus response to salinity were based on visual leaf injury and correlations with leaf chloride content. There were no root system observations recorded and no detailed physiological studies conducted. Such observations and measurements of physiological responses are necessary for a complete understanding of salt injury and tolerance in plants. This information is particularly valuable for efficient breeding and screening of new germplasm for salt tolerance. The objectives of this research are the following: 1. To compare the salt tolerance of citrus rootstocks commercially important in Florida and to determine which rootstocks are salt excluders or salt accumulators. 2. To determine the salt concentrations at which growth is depressed, water balance is disturbed, and leaves are injured. 3. To separate specific ion effects of salts from their osmotic effects by comparing growth, water relations, and plant chemical analyses under NaCl and PEG stresses. 4. To measure the effects of several NaCl and PEG concentrations on root growth and distribution. 5. To study citrus growth and water relations under non-uniform salinity (split-root system). 6. To examine the importance of calcium in reducing NaCl damage. LITERATURE REVIEW Salts Many hectares of land throughout the world are too saline for profitable agriculture (Carter, 1975). Large amounts of arable lands are being removed from crop production every year due to increasing soil salinity (Chapman, 1975; Epstein et al., 1980). Saline irrigation water combined with fertilizer application are the factors most responsible for increasing soil salinity (Epstein et al., 1980; Jones et al., 1952; Stewart et al., 1977). The ions in soil waters which contribute significantly to salinity problems are principalis sodium, chloride, calcium, magnesium, sulfate, potassium, bicarbonate, carbonate, nitrate, and occasionally borate ions (Bernstein and Hayward, 1958; Peck, 1975; Shainberg, 1975). However, most salinity research has involved NaCl because it is the most common salt in saline soils and irrigation waters. Mechanisms of Salt Tolerance in Plants Salt-tolerant plants are generally thought to be protected from salt stress by either ion accumulation or ion exclusion. Accumulation of high concentrations of ions in halophyte leaves has been known to be a salt tolerance mechanism (Flowers et al., 1977; Greenway and Munns, 1980). Salts can be tolerated because ions are compartmentalized in the vacuole and not in the cytoplasm. Hence, metabolic processes are not inhibited. These ions in the vacuoles balanced with neutral organic solutes in the cytoplasm lower the leaf osmotic potential. This allows 3 4 the plant to extract water from saline solutions. However, salt tolerance in glycophytes (nonhalophy tes) is related to ion exclusion because of the plant's inability to compartmentalize toxic ions in a useful way and to adjust osmotically (Greenway and Munns, 1980). Mechanisms of Salt Injury Salt damage to plants is caused by the decrease in the water potential of the soil solution or by the toxicity of specific ions. Some workers attribute most of the salt damage to osmotic stress (Bernstein, 1961, 1963; Bernstein and Hayward, 1958; Bielorai et al., 1978, 1983; Bohn et al., 1979). Others favor the idea that toxic effects of specific ions predominate in restricting growth and yields (Babaeva et al., 1968; Gollek, 1973; Strogonov, 1964). A common method of distinguishing between osmotic and ion toxic effects of salts is to compare the effects of isosmotic solutions of the salt with those of non-toxic organic substances. If the salt injury is simply osmotic, all solutes should produce the same injury at the same osmotic potential (Levitt, 1980). Polyethylene glycol (PEG), a non-ionic compound, has been successfully used as an osmoticum for subjecting plants and plant tissues to known levels of water stress (Janes, 1966; Kaufmann and Eckard, 1971; Kawasaki et al., 1983a, b). Osmotic Effect Water is osmotically more difficult to extract from saline solutions. Pair et al. (1975) pointed out that the addition of 0.4% salts had the effect of reducing the total available water in the soil by approximately 33%. Salt addition is analogous to soil drying since both result in reduced water uptake. In extreme circumstances, salinity can prevent water uptake even when the soil is at field capacity (Hartz, 1984). Water uptake by mature grapefruit trees, mature Valencia orange 5 trees, and Valencia orange seedlings was reduced as salinity increased (Bielorai et al., 1983; Hayward and Blair, 1942; Plessis, 1985). Ion Toxic Effect Ion toxic effect of salt is attributed to excess accumulation of certain ions in plant tissues and to nutritional imbalances caused by such ions. Ion excess has been defined as a condition where high internal ion concentrations reduced growth (Greenway and Munns, 1980). In many crops, salt injury increases with increased salt uptake. Raspberries were found to accumulate chloride ions more rapidly and consequently were more severely injured than blackberry (Ehlig, 1964). Tagawa and Ishizaka (1963) found that the primary cause of injury to rice by NaCl was chloride accumulation in the shoots. When treated with NaCl, a less resistant barley variety accumulated higher levels of chloride and sodium than a more resistant variety (Greenway, 1962). Salt damage to citrus has been mainly attributed to excessive accumulation of chloride and sodium in the leaves (Abdel-Messih et al., 1979; Chapman et al., 1969; Cooper, 1961; Cooper et al., 1951; 1952b; Cooper and Peynado, 1953; El-Azab et al., 1973; Furr and Ream, 1968; Grieve and Walker, 1983). Goell (1969) suggested that salt ions such as chloride in citrus leaves might shorten the life span of leaves by increasing chlorosis (loss of chlorophyll and photosynthetic potential) and by promoting senescence and abscission. Sulfate and other ions also caused damage to citrus (Bhambota and Kanwar, 1970; Bingham et al., 1973; Cerda et al. 1979; Hewitt and Furr, 1965a; Peynado and Young, 1964). It has been suggested that the accumulation of ions in large amounts in the leaves is the main factor causing leaf burn and inhibition of certain metabolic processes. 6 Sodium can also cause injury to plants through its deleterious effect on the soil. When the proportion of exchangeable sodium is relatively high, clay particles in the soil tend to disperse and -block the pores through which water flows. This phenomenon decreases the hydraulic conductivity of the soil (Bohn et al., 1979; Shainberg, 1975) and causes poor aeration. Studies by Aldrich et al. (1945) demonstrated that inferior performance of orange trees was caused primarily by poor water penetration resulting from sodium accumulation on the exchange complex. Nutritional Imbalance Salt can also damage plants by causing nutritional imbalances. High sodium levels can lead to calcium and magnesium deficiencies (Bohn et al., 1979). In spinach and lettuce, sodium salts decreased dry matter production as well as leaf potassium, magnesium, and calcium contents (Matar et al., 1975). Pumpkin and sweet clover plants subjected to NaCl showed potassium deficiency (Solov'ev, 1969). A decrease in potassium uptake at higher concentrations of sodium was found in sugarcane (Nimbalker and Joshi, 1975) and rice (Paricha et al., 1975). With increased salinity, potassium and phosphorus uptake decreased in grapes, guava, and olive plants (Taha et al., 1972), in wheat (Sharma and Lai, 1975), and in barley (Kawasaki et al., 1983b). In citrus, nutritional imbalance has been also attributed to depressed absorption of some nutrients. A decrease in the concentration of calcium, magnesium, and sometimes potassium was found when salt concentration in the irrigation water was increased (Jones et al., 1957; Patil and Bhambota, 1980; Pearson et al., 1957). 7 Plant Responses to Salinity Salinity has been known to adversely affect all stages of plant development such as germination, vegetative growth, and fruiting. Salinity has also been found to depress chlorophyll content, photosynthesis, stomatal conductance, root conductivity, and transpiration of many crops. For example, growth of citrus (Furr and Ream, 1968), Vicia faba (Helal and Mengel, 1981), pepper (Hoffman et al., 1980), alfalfa (Keck et al., 1984), bean (Meiri and Poljakoff-Mayber, 1970), and corn (Siegal et al., 1980) was significantly depressed under saline conditions. Yield of grapefruit (Bielorai and Levy, 1971; Bielorai et al., 1978, 1983), orange (Bingham et al., 1973, 1974; Chapman et al., 1969), celery (Francois and West, 1982), and muskmelon (Shannon and Francois , 1978) was severely reduced due to salinity stress. Salinity was found to alter fruit quality by decreasing the "pack out" of oranges at a commercial packing shed (Bingham et al., 1974) and by decreasing the marketable yield of tomato and melon (Mizrahi and Pasternak, 1985; Shannon and Francois, 1978). It was found that the relative amount of the premium grade fruit decreased with use of saline water even though there was a trend toward higher soluble solids and better taste (Bingham et al., 1974; Mizrahi and Pasternak, 1985; Shannon and Francois, 1978). Salinity reduced leaf chlorophyll content in grapevine, bean, barley, citrus and mangrove (Downton and Millhouse, 1985), spinach (Downton et al., 1985), and Acacia saligna (Shaybany and Kashirad, 1978). Leaf chlorophyll content declined only when certain amounts of salt ions accumulated in the leaves. Salinity reduced photosynthesis in spinach (Downton et al., 1985), rice (Flowers et al., 1985), Xanthium strumarium (Schwarz and Gale, 1983), beans (Seemann and Critchley, 8 1985), and Acacia saligna (Shaybany and Kashirad, 1978). Under most circumstances, photosynthetic reduction was attributed to ion accumulation in the leaves and to reduction in stomatal conductance. Salinity was found to reduce root conductivity in white lupin (Munns and Passioura, 1984) and beans (O'Leary, 1969; 1974). However, salinity did not affect root conductivity in barley (Munns and Passioura, 1984), sunflower and tomato plants (Shalhevet et al., 1976). Reduced hydraulic conductivity of roots has been attributed to root suberization and to reduced root membrane permeability. Salinity and Citrus Citrus is generally classified as a salt sensitive crop because physiological disturbances and growth and fruit yield reductions can occur at relatively low salinity levels (Bernstein, 1969; Bielorai et al., 1978, 1983; Boaz, 1978; Cherif et al., 1982; Cooper and Shull, 1953; Francois and Clark, 1980; Furr e< al., 1963; Kirkpatrick and Bitters, 1969; Marsh, 1973; Patil and Bhambota, 1980; Pehrson et al., 1985; Walker et al., 1982). Citrus Salinity Research The response of citrus to salinity is a topic of concern in many regions where citrus is grown especially the United States, Israel, Egypt, India, Spain, and Tunisia. In the United States, salinity studies essentially began in Texas during the 1940s. Investigations were led by U.C. Cooper with emphasis on differences in salinity tolerance among citrus rootstocks (Cooper, 1948; Cooper and Gorton, 1952; Cooper and Peynado, 1959; Cooper and Shull, 1953; Cooper et al., 1951). The work was conducted mostly on young budded trees grown in the field. Salinity treatments consisted of NaCl + CaCl added to Rio Grande river water. These studies led to the observation that chloride 9 exclusion was strongly correlated with salt tolerance. Chloride accumulation or exclusion and leaf injury symptoms were used to classify salt tolerant and salt sensitive rootstocks. Salinity studies on citrus were started in California in the 1950's (Harding et al., 1958a; Janes et al., 1952; Pearson and Goss, 1953), in Israel in the 1970s (Bielorai et al., 1973; Heller et al., 1973), and in Australia in the 1970s (Cole and Till, 1977). Most of these studies were conducted in the field on mature citrus trees and were focused on yield reduction and fruit quality alteration as a function of salt concentration in irrigation waters (Bielorai et al., 1978, 1983; Bingham et al., 1973, 1974; Boaz, 1978; Francois and Clark, 1980; Levy et al., 1979; Pehrson et al., 1985; Shalhevet et al., 1974). Recent salinity work in Israel was directed to plant breeding using cell culture techniques (Ben-Hayyim and Kochba, 1983; Ben-Hayyim et al., 1985). Recent work in Australia was conducted mainly with young rootstock seedlings grown in pots under glasshouse conditions (Behboudian et al., 1986; Grieve and Walker, 1983: Walker and Douglas, 1983; Walker et al., 1982, 1983, 1984, 1986). Salinity treatments consisted of NaCl added to a nutrient solution. These studies were focused on sodium and chloride exclusions mechanisms, water relations, and photosynthesis. Photosynthesis was severely reduced and photosynthetic reduction was attributed to a loss of turgor in salt excluder rootstocks and to chloride accumulation in salt accumulator rootstocks. Some salinity work on citrus conducted in Egypt (Abdel-Messih et al., 1979; Minessy et al., 1973), India (Bhambota and Kanwar, 1970; Patil and Bhambota, 1980), Spain (Cerda et al., 1979; Guillen et al., 1978), and Tunisia (Cherif et al., 1981; 1982; Zid, 1975; Zid and 10 Grignon, 1985, 1986) on budded trees and rootstock seedlings involved ion analysis and nutrient absorption. These studies showed that salinity caused nutritional imbalance, growth reduction, and leaf- burn. Growth reduction was attributed to potassium deficiency and foliar necrosis to sodium accumulation in the leaves. Salinity is of increasing concern in Florida. Salt water intrusion into groundwater in areas where citrus is grown has increased the need for salinity studies in Florida. Many citrus rootstocks are being used in Florida such as sour orange, Swingle citrumelo, Carrizo citrange, and Milam without knowing their salt tolerance. As a result, there is an incentive to study the salinity tolerance of these and other rootstocks which are commercially important. Physiologists often concentrate on the activities of shoots and neglect roots because they are out of sight and more difficult to study than shoots (Kramer, 1983). Roots play an important role in the growth and development of the entire plant. Their health, vigor and activity can be an index of the functioning of the above-ground parts (Crider, 1927). It is important to investigate root growth and distribution because roots are directly in contact with salts in the soil. Detailed information on the growth behavior and morphological development of citrus root systems under salt conditions is not available. The two major resistances to water movement through the soil-plant-atmosphere continuum are the roots and the stomata (Kramer, 1969; Kriedemann and Barrs, 1981). Root conductivity and stomatal conductance are important variables to be monitored in salinity studies because they can provide information on the water balance disturbance caused by salt. Root conductivity of some in citrus rootstocks under salinity stress has not been previously studied. Furthermore, 11 information relating root conductivity to stomatal conductance and transpiration as a function of different osmotic concentrations is lacking. Under field conditions, the roots of an individual plant grow in soil which varies in water content and salt concentration both in space and with time. In assessing the suitability of water for irrigation, it is usually assumed that plants respond to the mean root zone salinity (Shalhevet and Bernstein, 1968). However, some workers suggest that the least saline part of the rooting zone controls the overall plant growth and yield (Lunin and Gallatin, 1965). Responses of citrus to non-uniform salinity or to zonal salinization are not known. Citrus Tolerance to Salinity Scion. Differences in salt tolerance among citrus varieties or scions have been shown. Boaz (1978) concluded that Valencia orange had a lower tolerance to salinity than grapefruit on sweet orange rootstock. Bernstein (1969) reported that lemon was more sensitive to salinity than orange which was more sensitive than grapefruit. Miwa et al. (1957) also found that lemon was the most susceptible variety to foliar spray injury from sea water. Results of Pearson and Huberty (1959) showed that navel orange trees were more sensitive to irrigation water quality than Valencia orange trees. Budded on rough lemon, salt tolerance decreased in the following order: Hamlin, Valencia, Pineapple and Blood red sweet orange (Bhambota and Kanwar, 1969). Valencia seemed to be more sensitive to salinity than Shamouti when both were grafted on sour orange rootstock (Shalhevet et al., 1974). Rootstock. Some studies have indicated a wide range in salt tolerance among citrus rootstocks (Cooper, 1948; Cooper and Edwards; 1950; Coopei et al., 1952b, 1958). Cooper et al. (1951) found that 12 Cleopatra mandarin and Rangpur lime are relatively salt-tolerant rootstocks. They classified sour orange, rough lemon, sweet lemon, tangelo and sweet lime as sensitive rootstocks and Florida sweet .orange and trifoliate orange as very sensitive. In another study, Cleopatra mandarin and Rangpur lime were also found to be the most tolerant rootstocks and Carrizo citrange was the most sensitive rootstock (Joolka and Singh, 1979; Patil and Bhambota, 1978). Trifoliate orange and rough lemon were found to be very salt sensitive (Bhambota and Kanwar, 1969). Although some selections of sour orange differed in salt tolerance, Ream and Furr (1976) found that none of them was as salt tolerant as Cleopatra mandarin. Salt Exclusion. Exclusion of certain ions has been demonstrated in some citrus rootstocks. Rangpur lime and Cleopatra mandarin appear to be chloride excluders (Cooper, 1961; Cooper and Gorton, 1952; Cooper and Peynado, 1959; Douglas and Walker, 1983; Grieve and Walker, 1983; Hewitt and Furr, 1965b; Walker, 1986; Walker et al., 1983; Wutscher et al., 1973). Trifoliate orange appears to be a sodium excluder (Elgazzar et al., 1965; Grieve and Walker, 1983; Walker, 1986) and Citrus macrophylla a boron excluder (Cooper and Peynado, 1959; Embleton et al. 1962). This suggests the existence of a blocking mechanism in the transport of these ions (Fernandez et al., 1977). It also indicates the existence of apparently separate mechanisms which regulate the uptake and transport of ions (chloride and sodium) in salt-stressed citrus (Fernandez et al., 1977; Grieve and Walker, 1983; Walker et al., 1983). Ion concentration. Citrus is a nonhalophyte, and its tolerance to salinity is correlated with its ability to restrict the entry of ions into the shoots (Greenway and Munns, 1980). Injury to citrus from NaCl has been attributed to excess chloride accumulation (Ben-Hayyim and 13 Kochba, 1983; Cooper, 1961; Cooper and Gorton, 1952; Furr and Ream, 1969). In an effort to screen young citrus trees for salt tolerance, Hewitt et al. (1964) found that the leaves could be analyzed for chloride after 3 to 4 weeks of treatment with highly saline irrigation water. Fernandez et al. (1977) considered foliar chloride content as a suitable index of the soil salinity status and toxicity levels. However, Ben-Hayyim et al. (1985) showed the difficulty in determining if any particular ion could serve as a reliable marker for salt tolerance in citrus. Citrus Responses to Saline Conditions Photosynthesis. Photosynthetic rates were reduced by 50 to 75% after 70 days of NaCl stress (Behboudian et al., 1986; Walker et al., 1982). A decrease in photosynthesis is often caused by a drop in leaf turgor, but studies have shown different turgor responses to salinity. In one study with Rangpur lime, photosynthesis reduction was attributed to low turgor pressures in rangpur lime and not to leaf chloride or sodium concentrations since there was no significant difference in concentrations of these ions between salt-stressed and control leaves. In contrast to Rangpur lime, photosynthetic reduction during salt treatment in Etrog citron was associated with a marked increase in leaf chloride since turgor was not reduced. Their work established that a plant's capacity for salt exclusion alone or turgor maintenance alone was unable to protect citrus seedlings against photosynthetic reduction. Therefore, to improve salt tolerance in citrus, studies need to be focused not only on salt exclusing rootstocks but also on the ability of scions to maintain turgor. It appears that the inability to osmotically adjust and exclude toxic ions is related to citrus sensitivity to salinity (Zid and Grignon, 1986). 14 Yield. Citrus yield has been related to salt concentration in the soil (Bielorai et al., 1978; Harding et al., 1958b). According to Boaz (1978) and Maas and Hoffman (1977), the threshold salinity is an • electrical conductivity of the soil saturation extract of 1.8 dS/m (1.8 mmhos/cm) for oranges and grapefruit. Above this threshold, yield is reduced at a rate of 16% per dS/m. Pehrson et al. (1985) stated that 10 and 50% yield reductions for citrus were associated with electrical conductivities of the soil saturation extract of 2.3 and 4.8 dS/m, respectively. Salinity was found to significantly reduce citrus yield without visual symptoms (Pehrson et al., 1985). The use of moderately saline irrigation water (2.5 dS/m) decreased orange yield by about 30% without any visible leaf injury symptoms (Bingham et al., 1974). Within a concentration range of 2 to 2.7 dS/m, 9 to 18% yield reduction in grapefruit occurred without apparent tonicity symptoms (Bielorai et al., 1978, 1983). When irrigated with moderately saline water (15 to 30 mM, CaCl + Na,S0 + MgS04 ) , Valencia orange had yield reductions of 34 to 54% with no visible leaf injury symptoms (Francois and Clark, 1980). Leaf injury. Salinity effects develop slowly so that leaf injury symptoms appear after a certain period of time. However, the length of this time period is shortened by higher salt concentrations. Grown in the field, two-year-old Ruby red grapefruit on sour orange rootstock irrigated with salt solutions of 2500 mg/L (50:50 NaCl and CaCl2) showed no visible symptoms of salt injury during a one year period. Trees irrigated with 4000 mg/L salt solution developed leaf bronzing within 1 month and marginal burning of the leaves within 2 months. Trees irrigated with 5000 mg/L salt solution were completely defoliated within a one year period (Cooper, 1961; Cooper et al., 1952a). 15 Salinity and high water table. Relatively few studies have been conducted to investigate the effects of a combination of water table and salinity on citrus even though this condition exists in many part-s of the world. Studying the effects of salinity and water table on the growth and mineral composition of young grapefruit trees, Pearson and Goss (1953) found that the rates of defoliation and twig dieback due to salinity were greatly accelerated by a frequently fluctuating water table. In a more detailed report of the same study, Pearson et al. (1957) concluded that the salinity factor accounted for approximately 90% of the variance in growth while the water table factor accounted for only about AX. They found that sodium and chloride accumulated in toxic amounts in the leaves and were responsible for the decrease in growth. However, while investigating the effect of different water table depths and salinity levels on sweet orange, Kanwar and Bhambota (1969) observed that the adverse effect of water table was more pronounced than that of salinity. Both studies agreed that the interaction of water table and salinity affected the trees more severely than either condition alone. The fact that Cleopatra mandarin is more sensitive to flooding (Ford, 1964) but more salt tolerant (Cooper et al., 1951) than sour orange raises the question about the performance of these two rootstocks under saline conditions associated with high water table or flooding problems. Irrigation. Citrus is relatively sensitive to salinity, but can withstand high salt concentrations depending on the variety, rootstock, and irrigation management. Good irrigation management should consider the salinity factor in the irrigation water, in the soil, and in the root zone (Boaz, 1978). Methods of irrigation scheduling which do not account for salinity are not sufficiently accurate for scheduling 16 irrigation in areas with a saline high water table. Irrigation water containing about 250 mg chloride per liter reduced grapefruit yield by 28 to 322 when trees were irrigated at intervals of 40 days compared to intervals of 18 days (Bielorai and Levy, 1971; Bielorai et al., 1973). These studies demonstrated that the effect of salinity is more severe at lower soil water content. Overhead sprinkler irrigation should be avoided when using water containing high levels of salts because salt residues can accumulate on the foliage and seriously injure plants. Navel orange accumulated injurious amounts of chloride and sodium from sprinkler-applied water having 500 to 900 ppm total dissolved solids (Harding et al., 1958a). Considerable leaf burn and defoliation of these trees were found to be correlated with excessive amounts of sodium and chloride and lower amounts of potassium in the leaves. Leaf injury of navel orange trees developed at concentrations of 5 to 10 mmol/L of NaCl, CaCl2 or Na2S04 in the sprinkler-applied waters (Ehlig and Bernstein, 1959). Salt content of up to 1300 mg/L caused defoliation of sprinkler-irrigated citrus trees in Texas (Lyons, 1977). In Australia, during periods of high salinity in the irrigation water, foliar absorption of sodium and chloride occurred when using overhead sprinklers on citrus. It was believed that this problem caused poor tree health, low yield, and possibly poor fruit quality in citrus (Cole and Till, 1977). Frequency rather than duration of sprinkler irrigation is perhaps more important in foliar absorption of salts. Salt injury was higher under higher evaporation conditions and with short and frequent periods of overhead sprinkling (Eaton and Harding, 1959; Ehlig and Bernstein, 1959; Harding et al., 1958a). 17 Micro-irrigation is gaining in popularity not only in arid regions but also in humid subtropical areas. Micro-irrigation refers to both drip and microsprinkler irrigation. Micro-irrigation enables the use of poorer quality water that cannot be tolerated with overhead sprinklers. Direct foliar uptake of salts, and hence leaf injury, is avoided with drip irrigation (Calvert and Reitz, 1966). Nevertheless, saline water cannot be used indiscriminately with micro-irrigation systems. Comparative studies between overhead sprinklers and drip systems using saline water showed that vegetative growth, root development, and yield were greater with drip than with sprinkler irrigation (Goldberg and Shmueli, 1971; Shmueli and Goldberg, 1971). In a comparison of flood and drip systems, water high in chloride and boron was applied to young grapefruit trees on many rootstocks (Vutscher et al., 1973). More chloride and boron accumulation was found in trees that were flood irrigated than in thoc that were micro-irrigated. Drip irrigation at frequent intervals maintains a low soil water tension and prevents salt accumulation within the wetted zone. Consequently, water with higher salinity levels may be used without significantly affecting the yield. Nevertheless, salt accumulation under drip irrigation must be considered because salts may accumulate both at the periphery of the wetted zone and on the soil surface (Bielorai, 1977, 1985; Goldberg et al., 1976; Hoffman et al., 1985; Yaron et al., 1973). Reducing Salt Damage Role of Calcium Calcium has been known to have an ameliorating effect on the growth of plants under saline conditions (Deo and Kanwar, 1969; Epstein, 1972; Hyder and Greenway, 1965). This effect has been attributed to calcium 18 preventing the uptake of the sodium ion to injurious levels, and allowing the uptake of potassium (Uaisel, 1962). In the presence of adequate concentrations of calcium, bean plants were able to exclude sodium and to withstand the effects of relatively high NaCl concentrations (LaHaye and Epstein, 1969, 1971). In barley, inhibition of the absorption and translocation of potassium and phosphorus by NaCl was found to recover dramatically in the presence of calcium (Kawasaki et al., 1983b). Application of gypsum to the soil or in the irrigation water markedly reduced the percentage of soluble sodium in the soil (Harding et at., 1958b) and reduced the percentage of sodium in citrus leaves and roots (Jones et al., 1952; Pearson and Huberty, 1959). Calcium amendments are commonly used for replacement of exchangeable sodium (Richards, 1954). Calcium can flocculate soil in which clay particles and aggregates have been dispersed by sodium. Salt-affected soils can therefore be made productive by chemical amendment, drainage, and irrigation with high quality water, but sometimes the cost of these operations exceeds the expected returns from the land. Genetic Improvement In recent years, adapting plants to saline environments through breeding and genetic manipulation have been attempted (Epstein et al., 1980). The genetic basis for salt tolerance, using information from studies with whole plants, has allowed the identification of plants with increased salt tolerance. Another approach is to increase salt tolerance through cell culture (Croughan et al., 1981). In some species, the variability in salt tolerance may not be adequate for a successful breeding program because it may not be possible to find salt- tolerant wild relatives and use them as sources of germplasm. Suspension of cells from salt-sensitive plants in solutions 19 having various degrees of osmotic stress was found to be a promising technique to select salt-tolerant cells from salt-sensitive cells. This implies that the genetic information for growth in a saline environment may be present in salt-sensitive cells but is not expressed. Selection of salt-tolerant cells may provide genetic material that will help improve our understanding of salinity resistance at the cellular level. MATERIALS AND METHODS General Procedures This study consisted of 5 experiments involving citrus seedlings grown in greenhouses in central Florida. Seeds were sown in plastic trays composed of individual cells. The trays were filled with PROMIX BX [60% Canadian peat, 20% perlite, and 20% vermiculite with dolomitic limestone, superphosphate, calcium nitrate and fritted trace elements added]. The seeds were irrigated with tap water twice a week until emergence. Seedlings were irrigated with tap water every other day and fertilized with 20-20-20 (N,P,K) fertilizer once a week. The temperature and relative humidity in the greenhouses were controlled by both heating and evaporative cooling systems with conventional end-wall air circulation fans. The minimum and maximum temperature and relative humidity ranged from 20 to 35°C and from 40 to 100%, respectively. Three to 6 months after emergence, uniform seedlings were selected and transplanted into pots or wooden boxes containing fine sand taken from the top 30 cm of a citrus orchard soil. The soil was Astatula fine sand (hyperthermic, uncoated Typic Quartzipsamments) with a pH of 6.5 and a field capacity and a wilting percentage of 7.2% and 1.2% (volume basis), respectively. Seedlings were irrigated every 2 to 3 days with half strength Hoagland's solution #1 (Hoagland and Arnon, 1950) for at least one month before starting salt and polyethylene glycol (PEG) treatments. Treatments were started by adding NaCl, PEG, or other salts to the Hoagland solution. 20 21 The water holding capacity of the soil in the containers was about 18£ (volume basis). The irrigation frequency was 2 to 3 days. The amount of solution added each time was based on bringing the soil to slightly more than the water holding capacity of the soil in the containers to prevent salt accumulation in the growth medium and to prevent plants from undergoing a drought stress. Standard curves (Fig. 1, 2) of osmotic potential versus solute concentration were developed for NaCl and PEG 4000 by measuring vapor pressure and freezing point depression. The values obtained were similar to those of Steuter et al. (1981) who compared freezing point depression and vapor pressure methods for determination of water potential of PEG solutions. Electrical conductivities of the different treatments were determined with a conductivity meter. Electrical conductivity values were converted to TDS (Richards, 1954). Sodium chloride and PEG treatments were continued for at least 4 months, after which the plants were harvested and the roots were washed briefly with tap water to free them of sand particles. Shoots were separated into stems and leaves, and roots were separated into taproots, lateral roots, and fibrous roots (roots less than 2 mm in diameter). The material was oven-dried for 3 days at 60°C, weighed, ground, and retained for ion analysis. Analysis of variance (F-test) was used to determine significant differences and Duncan's multiple range test was employed for mean comparison at P < 0.05. Experiment 1: Effects of NaCl and PEG on the Root Conductivity and Leaf Ion Content of Seedlings of 7 Citrus Rootstocks The objective of this experiment was to compare the growth, ion content, and water relations of 7 rootstocks treated with different 22 NaCI Concentration (g!_1) 2 4 6 8 Fig. 1. Osmotic potential versus NaCI concentration as determined by vapor pressure (VPD) and freezing point depression (FPD). 23 PEG Concentration (g L ' ) 50 100 150 200 Fig. 2. Osmotic potential versus PEG concentration as determined by vapor pressure (VPD) and freezing point depression (FPD). 24 levels of NaCl and PEG. On October 20, 1985, 5-month-old uniform seedlings of 7 rootstock cultivars were transplanted into 33 cm-tall black plastic pots containing about 2.2 L of fine sand. Rootstocks studied were the following: sour orange (Citrus aurantium), Cleopatra mandarin (C. reshni), Swingle citrumelo (C. paradisi x Poncirus trifoliata), Carrizo citrange (P. trifoliata x C. sinensis), rough lemon (C. jambhiri) , Milam (C. jambhiri variant) and trifoliate orange (P. trifoliata) . The plants were watered with a half strength Hoagland's solution and were grown with this control solution for 2 months. Sodium chloride and PEG treatments were started on December 19, 1985, and nutrient solutions for treated plants were identical to that of the control plants except for the addition of NaCl and PEG. Sodium chloride and PEG were added to the half strength Hoagland's solution to achieve final concentrations of -0.10, -0.20, and -0.35 MPa. The basic nutrient solution (control) had an osmotic potential (OP) of -0.05 MPa. Treatments were as follows: Treatment TDS 0P_ _ EC NaCl (mg L"1) (MPa) (d! 1. NS control : Hi Hoagl. sol. 550 2. NaCl (0.10) : 1.0 g NaCl/L Hi Hoagl. sol. 1600 3. PEG (0.10) : 55 g PEG/L Hi Hoagl. sol. 460 4. NaCl (0.20) : 2.2 g NaCl/L Hi Hoagl. sol. 3000 5. PEG (0.20) : 105 g PEG/L Hi Hoagl. sol. 400 6. NaCl (0.35) : 4.2 g NaCl/L Hi Hoagl. sol. 4900 7. PEG (0.35) : 144 g PEG/L Hi Hoagl. sol. 350 Plants were adjusted to their final NaCl and PEG concentrations through a progression of -0.10, -0.20, and -0.35 MPa solutions at 2-day intervals to avoid osmotic shock. Plants were then maintained at their (MPa) (dS ra"1) (mmol) -0.05 1.1 0 -0.10 3.1 17 -0.10 0.9 0 -0.20 5.4 38 -0.20 0.8 0 -0.35 8.8 72 -0.35 0.7 0 25 final osmotic levels for 5 months. The experimental unit was a single seedling arranged in a split plot with 4 replications. The 7 salt treatments were assigned to the main plots and the 7 rootstocks to the subplots. At the end of the experiment, root hydraulic properties were evaluated while in situ on 4 seedlings of each rootstock as previously described (Graham and Syvertsen, 1984, 1985; Levy et al., 1983; Syvertsen and Graham, 1985). Before measuring, the soil was wetted to field capacity to minimize possible differences in soil hydraulic conductivity and equilibrated to 25°C in the laboratory. Each pot and intact plant were placed in a pressure chamber. The stem was then cut 10 cm above the soil and the chamber was sealed around the cut stem. The pressure within the chamber was increased gradually to a constant value of 0.5 MPa. After an initial equilibration time of 10 minutes, the weight of the liquid r xuded from the cut end was measured at least 5 times at 1 minute intervals. Osmotic potential of the exudate was measured by a Wescor vapor pressure osmometer calibrated with NaCl solutions. Each root system was washed free of soil, and the total length of fibrous roots of each plant was determined by the line-intersect method (Tennant, 1975). Water flow per root system measured in this way included a soil conductivity component and was expressed as weight of exudate per unit time and pressure (ug s~ MPa~ ). Root conductivity for each rootstock was calculated by dividing the water flow by the total fibrous root length. Thus, the root conductivity was expressed in ug/s/MPa per meter of fibrous roots (ug m s MPa ). Prior to measuring root conductivity, the trunk circumference of each seedling was measured at a point 5 cm above the soil surface and 26 converted to stem cross sectional area. Dry weights of leaves, stems, fibrous roots, and tap roots were determined. Shoot root ratio and specific root weight (root weight per unit length) were calculated. Leaf chloride content was measured using a Buchler-Cotlove chloridometer after extracting the leaf samples with a nitric-acetic acid solution. Measurement of leaf Na, Ca, Mg, K, P, Zn, Mn, Cu, and Fe content was performed using an inductively coupled argon plasma spectrophotometer after a wet digestion of the samples in a nitric-perchloric acid mixture. Experiment 2: Water Relations of Sour Orange and Cleopatra Mandarin Seedlings under NaCl and PEG Stresses The objective of this experiment was to study the effects of NaCl and PEG on the root conductivity, plant growth, stomatal conductance, and chlorophyll content of seedlings of 2 rootstocks differing in chloride accumulation characteristics, sour orange and Cleopatra mandarin (Cooper et al., 1951). Six-month-old uniform seedlings of sour orange and Cleopatra mandarin were transplanted on November 13, 1985, into 19-cm tall black plastic pots containing 5.5 L of Astatula fine sand. Plants were then watered to excess every 2 to 3 days with half strength Hoagland's solution for one month before NaCl and PEG treatments were started. The treatments were the same as in Experiment 1. The treatments were replicated 7 times in a split plot design with 2 main plots (rootstocks) and 7 subplots (solutions). All variables measured in Experiment 1 with the exception of the chemical analysis were also measured similarly in this experiment. Seedling height from the soil surface to the terminal bud was measured every 2 weeks. Leaf conductance to water vapor was measured on abaxial leaf surfaces with a Li-cor 1600 steady state 27 porometer at 2-hour intervals from 0700 to 1700 hours for 3 consecutive days. After 5 months of NaCl and PEG treatments, two 1-cm diameter disks were removed from the central area of 2 mature leaves per seedling to determine leaf chlorophyll content using N, N-dimethyl formamide as a solvent (Moran and Porath, 1980; Syvertsen and Smith, 1984). Two millimeters of N, N-dimethyl formamide were placed in a small bottle and the 2 leaf disks which were removed from the same seedling were weighed and then immersed in the solvent. The bottles were firmly closed and stored in the dark in a freezer for 2 months. The bottles were then removed from the freezer and left in the dark to equilibrate to the temperature of the laboratory prior to spectrophotometer examination. One millimeter of the chlorophyll extract from each bottle was diluted with deionized water and examined by means of a scanning spectrophotometer. The optical density of the extract was measured at wavelengths of both 663 mu and 644 mu, and chlorophyll content was calculated following the equations used by Arnon (1949). Leaf chlorophyll content was expressed as mg of leaf chlorophyll per gram fresh weight. New shoot growth was determined by counting leaf number and measuring leaf area over a 3-month period. The plants were harvested after 6 months of NaCl and PEG treatments. Total leaf area was measured by a Li-cor leaf area meter. Fresh and dry weights of leaves were determined. Specific leaf weight (SLV), expressed on a fresh and dry weight basis per unit of leaf area, was calculated. Leaf succulence was expressed as grams of water per gram of leaf dry weight. 28 Experiment 3; Fibrous Root Density and Distribution of Sour Orange Seedlings under NaCl and PEG Stresses The objective of this experiment was to determine the effect of NaCl and PEG on the root growth and distribution of sour orange seedlings. Five-month-old seedlings were transplanted on October 1, 1985, into root boxes filled with Astatula fine sand. The root boxes were similar to those described by Bevington and Castle (1982, 1985). Each container consisted of one plexiglas sheet (6.4 mm thick) attached to the front of a wooden box. The plexiglas was covered with a removable metal shutter to exclude light. The internal dimensions of a root box were 87 cm high, 27 cm wide, and 5 cm thick. The viewing surface was 23 dm and the volume was about 11.5 L. Drainage was provided by 3 mesh-covered outlets in the bottom of the box. The boxes were vertically oriented. Seedlings were allowed to adjust in their containers for 2 months. During this period, they were watered every other day with half strength Hoagland's solution. Plants were then treated with 2 concentrations of NaCl and PEG (total osmotic potential equal to -0.12 and -0.24 MPa). The experimental design was a randomized complete block with 3 replications using a single seedling per box. Treatments were as follows: Treatment TDS OP EC NaCl 1. NS control 2. NaCl (0.12) 3. PEG (0.12) 4. NaCl (0.24) 5. PEG (0.24) (mg L ) (MPa) (dS m ) (mmol) % Hoagland's sol. 550 -0.05 1.1 0 1.1 g NaCl/L ¥2 Hoagl. sol. 1700 -0.12 3.3 19 60 g PEG/L V2 Hoagl. sol. 450 -0.12 0.9 0 2.8 g NaCl/L V? Hoagl. sol. 3300 -0.24 5.9 48 110 g PEG/L 'k Hoagl. sol. 390 -0.24 0.8 0 29 Root growth was recorded at 2-week intervals by using colored pencils to trace the root system onto transparent acetate sheets. Plant height was measured at 2-week intervals. Stomatal conductance was measured about every 2 weeks and for 2 consecutive days at 2-hour intervals from 0700 to 1700. After 6 months of NaCl and PEG treatments, the plants were taken from their boxes by removing the plexiglas wall and inserting a needle board to hold the root system in place. Leaves, stems, and roots were separated and roots were divided in place into 3 equal compartments (top, middle, and bottom). Shoot and root dry weight, shoot root ratio, leaf number, plant height, root length, specific root weight, and stomatal conductance were determined as described in Experiment 2. Experiment 4: Response of Split-Root Sour Orange Seedlings to Salinity The objective of this experiment was to determine and quantify the growth and water relations of sour orange seedlings when only a portion of the root system was exposed to NaCl or PEG. A split-root system was initiated using the technique of Koch and Johnson (1984). The tap root of each seedling at the 3-leaf stage was cut to a 1 cm length and all other roots were removed. The remaining portion of the tap root was dipped into a 50% ethanol solution containing 5 grams of IBA (indolebutyric acid) per liter. Seedlings were then placed in PR0MIX BX, watered daily and fertilized weekly for 2 months. Seedlings which had 2 uniform adventitious root systems were selected and transplanted when 5 months old into 2.2 L square plastic containers stapled together along one side (Fig. 3). These seedlings were left to adjust in their double pots for 1 month before NaCl and PEG treatments were imposed. The treatments were replicated 4 times in a randomized complete block design and are shown below: 30 Fig. 3. Sour orange seedlings with a split-root system. a. Root development after 2 months. b. Container system used to grow split-root seedlings. Treatment 1. NS/NS (no salt) 2. NS/NaCl (0.10) 3. NaCl (0.10)/NaCl (0.10) 4. NS/NaCl (0.20) 5. NaCl (0.20)/NaCl (0.20) 6. NS/NaCl (0.35) 7. NaCl (0.35)/NaCl (0.35) 8. NS/PEG (0.20) 9. PEG (0.20)/PEG (0.20) 31 TDS (mg L"1) 550/550 550/1600 1600/1600 550/3000 3000/3000 550/4900 4900/4900 550/400 OP (MPa) -0.05/-0.05 -0.05/-0.10 -0.10/-0.10 -0.05/-0.20 -0.20/-0.20 -0.05/-0.35 -0.35/-0.35 -0.05/-0.20 -0.20/-0.20 EC (dS m"1) L. 1/1.1 1.1/3.1 3.1/3.1 1.1/5.4 5.4/5.4 1.1/8.8 8.8/8.8 1.1/0.7 0.7/0.7 400/400 Water relations variables were monitored on 4 successive days during the fourth month of salt treatment. Leaf water potential was measured at sunrise and at midday on fully expanded leaves using a pressure chamber. Leaves were then removed from the chamber, trapped in double plastic bags and rapidly frozen at -20°C. Leaves were subsequently thawed after 48 hours and their osmotic potential was determined with a vapor pressure osmometer. Turgor potential was obtained by subtracting the osmotic potential value from the water potential value. Morning and midday stomatal conductance and leaf transpiration rates were measured with a steady state porometer. For anatomical study, 2 mature leaves per plant from NS/NS and NaCl (0.35)/NaCl (0.35) treatments were selected from about half-way between the first leaf and the shoot apex. Two small rectangles were cut at mid-lamina of each leaf, frozen immediately, and cut by a Cryostat minot rotary microtome in sections 10 microns thick. Sections were then thawed in a phosphate buffer saline solution. Twenty randomly selected leaf cross sections per treatment were fixed for a light microscopy study. 32 After 4 months of NaCl and PEG treatment, the plants were harvested, and shoot and root dry weights were determined. Experiment 5: Effects of Calcium on Sour Orange Seedlings Grown under Saline Conditions The objective of this experiment was to determine if the addition of calcium to saline irrigation water would reduce salt damage. Three-month-old sour orange seedlings were transplanted on August 10, 1986, into the same pots used in Experiment 2. Salt treatments (Table 1) were started after 1 month of adjustment, and seedlings were irrigated every 2 to 3 days for 4 months. The treatments were replicated 8 times in a randomized complete block design. The plants were watered the night before harvest and leaves were removed the following morning. Fresh and dry weights of leaves, stems, and roots were recorded. The succulence of new and old leaves was computed. The dried, mature, fully expanded leaves were ground and their mineral content was determined as in Experiment 1. 33 V) a. i-l X c w 1 B 1 4-> w nJ 4-J 01 c u a> *-> B 4-* A-J £)\£>C0 o o o o m o o o in o m co m m m m n * u cd u X S o CO m nl r-- o 00OOOOOOO ZH 0> 1-1 V) 0) 4-1 X o w ^v 1 bO W c o •H x: 4-* bO (4 •iH 1-1 CU 4-* > c o o o w dc a, (0 t/i 4-J O «J l-i •1-1 tH r-t o o c 14-1 o v-t X U 4J bO D O 4-. C c o nJ -i-i (0 rt x x 37 # 80 10 * 60 a: | 40 u. 5 20 2 o 80 2 60 >» 5 40 u 3 20 2 80 * 60 40 20 SO Q RL Ik SC £C nML „ so CM RL r-.CC ML nifhiimihny cc t>c sc PT CM ! I - "I SO n ML RL Rootstock Fig. 4. Effect of 3 NaCl concentrations (a = -0.10 MPa, b = -0.20 MPa, c = -0.35 MPa) on the total fibrous root length, root hydraulic conductivity, and water flow rate for seedlings of 7 citrus rootstocks. 6 *-> C bO -O o a; crt O W > B 1 cfl a> i XI - c *-> ej o -h O **H CO a> u o o a, X ► 0) u JS *-> • 1 •m co co i bOT3 C co C 3 o a. Oi X •h ac rH 01 •h in 4-» i-» x> O O O c • O O o o os ij O 1 o ac cd r^ c > M-t o 01 o C» »J~. >* *-> rH -*-» *J CO O • rH lt-1 -H o > O *-« a -H C *-> w cut c> hO *J SCO rH J3 •O'H a C r-t ■*-» bO o -o ci c C cO > a. o x *-> bO CO 3 •i-i CO > o, •H X J3 CO O rH bO c cd OS 4J C c o CO -rH 4-J 01 O CO c c (0 (0 01 01 x: x: N >, 41 50 60 70 Specific Root Wt (mg m 1 ) Fig. 5. Relationship between root hydraulic conductivity and specific root weight of seedlings of 7 citrus rootstocks under non-stressed conditions. 42 -a i-J o> c 01 o> CO E •rH c 1-1 •H cu i— i F XI +j m CO CU •rH u > 4-1 cd rd o> cu ►J J rd rd cu cu 4-1 14-1 rd rd cu cu rd rd cu cu U O X> 3 U XI -O JZ rd rd rd 01 01 u J J « rd rd cu cu bo rd 3 r-H O -rK OS ac o rd y-i X) 0) •n cu c -.-I u CU T3 3 4-» XI CU JZ r-t o M-l a. c td B cd cu o u JU« c Q. u O 3 I-i X) T3 "4-1 U-4 rd m r-l o U t-> bO C OJ x: w e bo u-i 4-> o rt u VI be c c a> •H o H c T3 o (V o 0) V\ o w 4-4 Oi o T3 c ^ rt ^ .-i 4-> C ) C (fl M a; td T3 0) c nJ w t-i D rt u •rl H i— 1 o O c 1M o ■t-l 1< u *-" o bn N c •rH (Tl lJ U 1-1 4-» m •H u O — 1 (Tl u e (U u wi o c o nj in o *-h o ^ o o o t— I '**' ^ v-' C C rt a) C J u o o 0) (U tfl LJ w w 3E s: 2 D-, Q- Q-, M >< 45 1.5 W c Cl, 01 *J TJ c c o «» u i-H a> o •X) m •H 2 u o bn r-v a; N c a, •H rcj M-l X U 1m o w M i a) •rH (0 1 O o bo w c c •H O rH -H o *0 4J V i-H C o c ■rt u u-j i-i ^r o - o O o a o a rH rH rH u u u tn (D (8 rt 2 2 2 2 bO C rd 05 rd rd w «J 3 -H l-i I— 1 H O a >4-l o •H o M Cu ■t-* 47 48 X 4-1 w o 1 w w u c c o •rH r-H -o m Q) M o l-i -o o ■w u c bo 0) G JZ ■H CO u D cu rH a. y-i X CxJ > 1 a; 1 c CO bn >% c r-i •H JZ .-< T1 c <» o cu sc CO c c cu (0 (0 > QJ o c TO O "O c o o ro E o CO Sour orange 20 10 \\NS x\NaCI(12) ^ ■NaCK.24) A ^°^0PEG(.12) '^.u— d— d— d P E G( . 2 4 ) 11 15 *t 1 1 15 Time ( hr) Fig. 10. Relationship of time of day to stomatal conductance of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG during 2 consecutive days. Measurements were started on April 17, 1986. Seedlings were irrigated the day before measurements were started and not irrigated until after measurements were completed on Day 2. 60 60 50 - o 40 en c5 30 - 20 - "O CD CD en 10 - Sour orange ^ NS . NaCK. 12) . NaCK. 24) / /-^" ^l PEG(12) ^ Jk>^ ^^ PEG(.24) 1 1 • Dec 4 Mar 6 TIME (date) Jul. 1 7 Fig. 11. Growth of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG. 61 Sour orange O) c Z 2 o NS • NaCI(.12) ■ NaCK.24) o PEG(12) a PEG(24) Dec. 4 Mar.6 TIME (date) Jul. 1 7 Fig. 12. Fibrous root length of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG. 62 5 o i- (D o o -C 10 6 - 40 30 2 20 CD o o a: 10 NaCK.12) NaCK.24) 0 PEGU 2) 0 PEG(.24) Dec Jan Feb Mar Apr TIME (month) May Fig. 13. Fluctuations in shoot and root growth of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with added NaCl or PEG. 63 u *-> (fl c tx a; E e o •r-l a 1-1 0) ro a X a; w J3 I i w C 4-< ■i-i c a> ^-x f= fc* ra T7 a) c u n) v-» *-" T5 bO C c m a> i— i f-i o IrJ m o z o u 4-> c to a» D u O a; M «-i XI 4-1 •i-t •iH tu T3 o o 1-1 4-? o o <-< o o t— 1 o o O o i-H ^ IK° CO ON a> -o o rsi CM CN) CM en •~> c c o (0 -rH .—I *-> O. rd u a <4-l a) o to c c '*:: ■ • •*- X ■/.■i; 1 \ ' i I )" ■-- J ' ■ ' • ■ ' j i :i '■(¥ * ■ . \ * ; . f f •. PEG(.24) Fig. 14. Root density and distribution of sour orange seedlings growing in root boxes under non-stressed (NS) and stressed (NaCl, PEG) conditions. NaCl and PEG treatments were at -0.24 MPa osmotic potential. 65 bottom section, only 5 to 16% of the roots developed in the stressed chambers as compared to 26% in the controls. Fibrous root length at the plexiglas face, measured from tracings made on acetate sheets with colored pencils, was compared to the total fibrous root length measured at the end of the experiment. Root length against the plexiglas represented 3 to 4%, 2 to 3%, and 4 to 5% of the total root length in the top, middle, and bottom of the root boxes, respectively. From the comparison of root lengths at the plexiglas and in the box, it was concluded that growth and distribution of citrus roots at the plexiglas-soil interface correlated satisfactorily with growth and distribution of roots in the bulk soil. Experiment 4: Response of Split-Root Sour Orange Seedlings to Salinity Uniform salinity was significantly more damaging to sour orange seedlings than non-uniform salinity (Table 16; Fig. 15). Shoot dry weight was reduced only slightly (9 to 21%) when half of the root ystem was irrigated with saline solutions. When both halves of the root system were irrigated with saline solutions, shoot dry weight was reduced 45 to 81% (Table 16). The trend was similar with root dry weight in that stressing one-half of the root system resulted in only a moderate reduction (16 to 31%) in root dry weight. Stressing both halves gave a much larger reduction in root dry weight (43 to 79%). In the split-root test, shoot growth did not correlate well with the average salt stress of the total root system. The average osmotic potential of the NS/NaCl (0.20) treatment was -0.12 MPa. Even though this was slightly greater than the average osmotic potential of the NaCl (0.10)/NaCl (0.10) treatment, shoot dry weight was 35% (10.7 g) less in the NaCl (0.10)/NaCl (0.10) treatment. Similarly, shoot dry weight in the NaCl (0.20)/NaCl (0.20) treatment was 50% (14.5 g) less than that in 66 > 4-* c >> a) u R T> ■rH U i-J tl> o a. o X u w -a | c to S a; 10 4-» t/i O a; O m -C *-• c/} to T-i -* cm m -O -o o U-l a TJ (fl O -O a; -O bo o bo rsl IN. CM 00 i-H .h m .h .H CM CNJ en CM O i-H CO v£> in «* m CI CM CM •H \ v \ N. "s \ N, N, ~* co ON U"> - U-l < o Cfl -D J3 U-l xi x: bo O JZ v£> si- rH CT> CT\ VO n o rH ~tf w C_> v^ U O to (0 (0 Cd >-^ 2 -H 2 rH 2 rH a> \ O \ u •x u \ o m m to m co nj to w 2 2 2 2 2 2 2 a. c •H X 4-> •H • > CO i-. c c o ni -«h rH *J a. aj c c to rt 67 NS/PEG(.20) PEG(.20) Fig. 15. Split-root treatment of sour orange seedlings under uniform and non-uniform NaCl and PEG stresses. NaCl treatments were at -0.10, -0.20, and -0.35 MPa osmotic potentials. PEG treatments were at -0.20 MPa osmotic potential. 68 the NS/NaCl (0.35) treatment, even though both of these treatments had the same average NaCl stress (-0.20 MPa). Under uniform salinity similar to Experiments 1, 2, and 3, shoot growth was more reduced than root growth. However, under non-uniform salinity, root dry weight on a percentage basis appeared to be more reduced than shoot dry weight (Table 16). Partial leaf burn occurred after 4 weeks in the NaCl (0.35)/NaCl (0.35) treatment and after 5 weeks in the NaCl (0.20)/NaCl (0.20) treatment. No leaf damage symptoms were noticed in the remaining treatments until the end of the experiment. Water relations variables were monitored on 4 successive days during the fourth month of salt treatment. Data were combined because no significant differences were found from day to day. Similar to growth, water relations variables were also significantly more disturbed under uniform salinity than under non-uniform salinity conditions. With uniform salinity, leaf water and turgor potentials decreased significantly from morning to midday, but leaf osmotic potential did not (Fig. 16). Leaf water potential, osmotic potential, stomatal conductance, and transpiration decreased with increasing NaCl and PEG concentrations in the irrigation water (Tables 17, 18). Turgor potential significantly increased in response to NaCl treatments particularly during the morning. A significant positive correlation was found between stomatal conductance and transpiration (Fig. 17). Similar to findings of the preceding experiments, PEG at -0.20 MPa was more damaging than NaCl at the same osmotic potential. Cross sections of leaves from control (NS/NS) and from NaCl (0.35)/NaCl (0.35) treatments, compared by light microscopy, showed that the number of cell layers in the epidermis, the palisade, and the spongy 69 03 CL 5 1.5 u 1.0 o ■5 °-5^ Midday ~_ -0.1 -0.2 -0.3 Salt Treatment (MPa) Fig. 16. Leaf water, osmotic, and turgor potential of sour orange seedlings irrigated with nutrient solution containing no salt (NS) or with NaCl added to both root halves. Solid figures are morning values and open figures are midday values. 70 *■> c a. e rfl w ■i-i I j-j i C w ai UJ w o w a- a; u u l-l o to rxi u C9 D W 4-" (X, T3 T3 C c BJ (0 .. .-H O O -rH m 4J EC O F u W 0) O T3 c o l-i a; V) 4-> bo cd c > •rH i-H >4-l •n rt <1> » a; nt cm TJ c ■n ro U r: o x: c oo oo oo oo bo C 05 o o m r-l CM en o o O O *-' ' — ' ^ CM rH 1—1 .—1 O u u u ^ ^n cd ^ rd ^-» (fl O 2 O 2 m 2 «u >-t \ cm \ m \ o w CM Oj o o o o o m • \ v.^,-1 wcm y_^m o o •—'CM -H O ■-H O i-< o u ^ U ^ u ^ U O nj Cd rfl W v^ 2 -H 2 rH 2 -H 0. N. O \ u ■v U ^ o en rt LO rci oo rd 00 w 2 z 2 2 2 2 2 0u • > w *•» c c o nj -h a. m c c rd n) a) a; 71 i-l to rt 00 u cu •rH u Du 1-J 00 to c tfl CJ u w »-' IX T3 X> C c rt rt rt xi X) V rt x> XI o XI o m m KD CO -3- ro f~~ 1-1 o o O O O O O O ^ nS m Z ^O ro \ o w . ^~. cm a. Z rH Cu rt rt \ U \ O CU dJ in ra 00 DJ z: s: 2; z Z Cm N >. 72 2.50 - w 2.00 - £ 1.50 - 1.00 - 0.50 - .20 .40 .60 .80 Stomatal Conductance (cm s ' ) Fig. 17. Relationship between transpiration and stomatal conductance of sour orange seedlings. 73 raesophyll in control leaves and NaCl-treated leaves were similar. Epidermal and palisade cells of the control and NaCl-grown leaves were also similar in size; however, the spongy mesophyll cells of the • NaCl-treated leaves were about 3 times larger than those of the control (Fig. 18). The overall increase in leaf thickness due to NaCl was relatively small (23%) because the enlarged cells of the spongy mesophyll were tightly packed with much less intercellular space. Cells of the spongy mesophyll in NaCl-treated leaves also had fewer chloroplasts than those in the control leaves. Experiment 5: Effects of Calcium on Sour Orange Seedlings Grown under Saline ConditionI Addition of NaCl to half strength Hoagland's solution significantly reduced growth of sour orange seedlings. Shoot, root, and total plant dry weights were reduced by about 30% (treatments 2 and 10) when 40 mM NaCl was added to the nutrient solution (Tables 19, 20). However, addition of 7.5 mM CaS04 (treatment 3) to the salty solution decreased the adverse effect of NaCl on growth. Furthermore, addition of only 5 mM CaSO (treatment 12) completely inhibited the adverse effect of NaCl. Addition of either KCl (treatments 6 and 7) or CaCl2 (treatments 5 and 8) to the salty solution did not improve plant growth. In the leaves of the sour orange seedlings, addition of NaCl to the nutrient solution significantly increased sodium and chloride, decreased calcium, magnesium, and potassium but had little or no effect on phosphorus, zinc, manganese, copper, and iron (Table 21). Sodium and chloride accumulation in the leaves usually reduces growth. Addition of CaS04 (treatments 3, 4, 11, and 12) to the saline solution reduced sodium and chloride content and, therefore, improved plant growth. Addition of KCl (treatment 6) did not reduce sodium and chloride; hence, 74 Fig. 18. Cross sections of sour orange leaves. a. Leaf cross section of non-stressed seedling. b. Leaf cross section of NaCl-stressed seedling, i.s. = intercellular space. 75 u to o rd rd tO U >n o On on O CM NO CO r^ o NO ON m CM CM CO CM CM CO CM i 01 T3 (U 13 XI O Q) T3 TJ Xl -a XI rd O rd X) -a O O CU rd o rd rd CM rH .-1 CM no st CJ> as •rH •rH rd rd :c E B ^ tvi B O O c_> U B rH i-H m m 3C s: m rd rd as 3C m CO r"* 6 e r-» o O B E r^ r-H CO r- r>- T-H o c O c rH in + + + + + + 1 1 + + ^ r-H r- 1 r-H r-H r— 1 r-H r-H ^ r-H r-H r-H to u U o O u U O to u O CJ> z rd rd rd rd rd rd rd Z rd rd rd z Z z z Z z z w Z z Z 1—1 CM rO cu E •>H 0) u U QJ c CX 1 O 1 a v\ a J-J to c a; u-i F (fl -t-» 01 m r— 1 OJ u ■w i-> c (0 *-> C M-l O C H 3 o CJ i-H X II ^-v AJ ai > o c >> a> u i-H T) D a bf) o V D u 00 d) 4-» bo — u co 4-J QJ Z > > o c >» r-f (d u X! 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(V R) ItJ . u O CM CM CM CM CM rH rH r-l o O in /-> ^v r-N o .— I CM CO o o m l-l rH CM CO rt XJ o O d cm c o O o x: o i u rH rH rH > — s u u c o o (J V3 (6 rt (0 u w w 2 2 2 2 a, Cu a, bo c CD OS nJ a) an je 100 bO C C CD •h e s-^ C bO O s-x •H 4-> t-1 rtl £i l-i bf 4-1 •H c <1> o c >, o u o T) C5 i-> w c a, K) r-1 "O a. c id r-\ fO rH 4-> u o Hi H 2 rt i/) 3 m u •ct rH I— I O o c <4-l o •H X U bO 3 O 06 101 CM t-H .-I w w ^ c c . *-• c c o 102 CO u bo V c ex •H X r- 1 w T> 1 CU 1 fl> CO CO a o y-i •rH o 4-> nj U 4-> H C a cv r-l &4 (0 C XI o c •H rtl 4-> CJ 1— I cu c_> CO rcl 2 to CO 4-= o c ft) CO 4-> 3 rt U •H rH I— 1 o o c VM o •r-1 x, M 4-J 0) c > to o x: cu a» c bf *s° r* (Tt CO c o x; 2 m rH 4J u H c o (0 o ID rH rH a; bo ~ c rH u > 4-J S) •rH U m tH CM CM d o CM «H rH CM r-t H c o rt fl o cu cu w a: x 103 O X u u 1 p^ 1 ^cn w K wo c ■u nj C rt o •H u 0) c c o (6 •<-< ^_< c c rt rt o V (V u X £ 104 ^ c a -H bo o CO u 3 F: 4-J (J) O 4-J D Z z bo c nj 04 CO 4-. c c c cy cu 105 o |HN bo bo *-> C rt •rH Ul c o o id O 2 w 4-> 3 c u 0) o In J2 QJ CLU-t o> a -J D nJ ij rt •H (TJ a o X T3 T3 ■o rH O rH co CO ON vO m m o T3 e T3 O Xi cd O X rt m i-H CTv .-1 CO !>■ rH -tf ^O •H O rH rH rH rH rH rH as O O o O o O O c O XI X td rt rt rd o a 00 o rH lO -3- m in r^ 4-> rH rH rH rH rH rH rH o O o o O O o O o f-l o rt X X n! 3 U O CTn CNl ■ CO ON CM rH CM rH rH C o o rH rH rH rH rH «) e o o O O O O o V X> X bO rt XI X (0 <« fO m c n) 0 CO co OS O O o u O o o O rH rH rH o o o o O O O O r-l o o in ^-> ^-^ *— * o .— i CNl CO o o in a u •—< CM CO 01 l~t d o o e c *. — ' ' — "* ^-^ o O O 4-» o m u H <—\ .— 1 01 u CJ o u o u u in rt (0 nj w u w H z z z Z Cu c D-, • > w J-> c c o I-H 4-> a. at c c rt rt 106 M 4-> o c o E 1-1 o 0) o D. u X W hn 4-< c rt H tj ^H 4-> •o C 0) a> f-H a C ) o id O 2 o 4-J c C -J T3 n) t/> 4-* 3 (fl l-i •H H i-( O o c >4-l o •H i, t-l 4-1 4J C c o (0 -r* 107 4_l rt c 0) i-t 4-> o c rt O 2 o 4-» 0) c CO HI a> u c V rt m-i W4-I c •rH rt T3 5 lJ U-l c to to 4-1 D rt U •H H r-l o O c 4-4 o •H x U *-• o M N c ■H rt u u 1-1 i-t m •H CJ O C rt o o Csl o s • > CO *j c c o i— i *-> a. ni m as x: x: 108 Table 31. Seedling height (cm) of seedlings of 2 rootstocks grown for 6 months under d concentrations — Experiment Lfferent NaCl 2. and PEG Sour orange Cleopatra mandarin Treatment X lower X lower (-MPa) Mean1 than NS Mean than NS NS control 104 ay 0 106 a 0 NaCl (0.10) 64 b 39 79 b 26 NaCl (0.20) 52 c 50 71 c 33 NaCl (0.35) 48 cd 54 55 d 48 PEG (0.10) 60 b 42 63 c 41 PEG (0.20) 41 de 61 54 d 49 PEG (0.35^ 38 e 64 43 e 59 Mean of 7 plants. Mean separation within columns by Duncan's Multiple Range Test, 0.05 level. 109 Table 32. Total leaf area (cm ) of seedlings of 2 rootstocks grown for 6 months under different NaCl and PEG concentrations — Experiment 2. Sour orange Treatment % lower (-MPa) Mean* than NS NS control 3595 ay 0 NaCl (0.10) 2002 b 44 NaCl (0.20) 1381 c 62 NaCl (0.35) 881 d 76 PEG (0.10) 1019 cd 72 PEG (0.20) 431 e 88 PEG (0.35) 213 e 94 Cleopatra mandarin % lower Mean than NS 3601 a 0 1782 b 51 1421 be 61 725 de 80 913 cd 75 498 de 86 294 e 92 Mean of 7 plants. yMean separation within columns by Duncan's Multiple Range Test, 0.05 level. 110 Table 33. Specific leaf weight (mg/cm ) of seedlings of 2 rootstocks grown for 6 months under different NaCl and PEG concentrations — Experiment 2.' Sour orange Cleopatra Fresh wt mandarin Fresh wt Dry wt Dry wt Treatment basis basis basis basis NS control 25.1 ay 9.0 b 18.7 b 8.3 c NaCl (0.10) 27.5 a 9.5 b 18.9 b 8.4 c NaCl (0.20) 27.9 a 9.6 b 19.1 b 8.5 c NaCl (0.35) 28.2 a 9.6 b 21.1 a 8.7 c PEG (0.10) 18.2 b 11.2 a 18.3 b 10.1 b PEG (0.20) 17.4 be 11.5 a 18.2 b 12.1 a PEG (0.35) 14.4 c 11.3 a 18.1 b 11.9 a zMean of 7 plants. yMean separation within columns by Duncan's Multiple Range Test, 0.05 level. Ill Rootstock CM SO SC CC RL ML PT